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− | <div class="title"> | + | <div class="title"> PETZAP: Integrating enzymatic PET degradation into Microbial Desalination Cell technology</div> |
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− | <div class="text"> | + | <div class="text"> |
+ | <b>Team members</b> | ||
+ | Olaide Ibiyemi, Juliette Champaud, Stefan Hristov, Pedro Lovatt Garcia, Daniel Castellano, Anna Su, Li Xu and Oliver Hernandez Fernandez | ||
+ | <br><br> | ||
+ | |||
+ | <b>Project Abstract</b><br> | ||
+ | The world’s oceans are suffocating from an annual addition of 8 million tons of plastic which threatens marine ecosystems and exacerbates water scarcity, affecting over 2 billion lives globally. Our project aims to tackle these two global challenges by integrating enzymatic polyethylene terephthalate (PET) degradation into a Microbial Desalination Cell (MDC). The system involves a 2-step process of co-culturing engineered E. coli to express a PETase-MHETase fusion degrading PET and P. putida to achieve further degradation and produce lactate, which then supports the biofilm growth of exoelectrogen, S. oneidensis, generating bioelectricity for desalination. Desalination efficiency was maximised by optimising lactate secretion, co-culture design, and MDC configuration based on the results from our flux balance analysis (FBA) and agent-based model, which simulateding bacterial plastic degradation and bioelectricity production, respectively. Our findings were combined with iInsights for further technical optimisation and feasible implementation at large scales, were obtained through iterative repeated engagement with experts and stakeholders. | ||
+ | </div> | ||
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− | + | <div class="section third" id="problem"> | |
− | <div class="section | + | <img src="https://static.igem.org/mediawiki/2020/4/4d/T--UCL--Poster_problem_section.png"> |
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<div class="info"> | <div class="info"> | ||
− | + | <div class="title"> | |
− | <div class="title"> | + | <h1>Problem</h1> |
− | < | + | </div> |
− | <div class="text"> | + | <div class="text"> |
+ | By 2050, it is estimated that plastic will outweigh all fish in the ocean (1). Plastic pollution of the Earth’s waters exacerbates freshwater scarcity with over 2 billion people living under high water stress (2). Recycling and desalination are current methods used to address these problems. However, only 14% of plastic packaging is recycled (1) and current methods of desalination such as reverse osmosis are highly energy-intensive and not environmentally friendly. As plastic is currently an integral part of modern life, we need to change how we see and handle end of life plastic, in a more environmental way. | ||
+ | <br> | ||
+ | <img src="https://static.igem.org/mediawiki/2020/a/ab/T--UCL--Poster_problem.png"> | ||
+ | </div> | ||
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</div> | </div> | ||
− | + | <div class="section third" id="motivation"> | |
− | + | <img src="https://static.igem.org/mediawiki/2020/c/cc/T--UCL--Poster_motivation_section.png"> | |
− | <div class="section third" id=" | + | |
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− | <img src="https://static.igem.org/mediawiki/2020/c/ | + | |
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<div class="info"> | <div class="info"> | ||
+ | <div class="title"> | ||
+ | <h1>Motivation</h1> | ||
+ | </div> | ||
+ | <div class="text"> | ||
+ | <img src="https://static.igem.org/mediawiki/2020/1/10/T--UCL--Poster_motivation_plastic_economy.png"> | ||
+ | <br> | ||
+ | 8 million tons of plastic waste are disposed in the ocean annually (3). With plastic being a common and non-degradable commodity, its linear life cycle is not sustainable for its use. Therefore, we were inspired by the possibility of synthetic biology to create a circular plastic economy by repurposing plastic waste as a resource for other uses. | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
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<div class="section third" id="idea"> | <div class="section third" id="idea"> | ||
− | + | <img src="https://static.igem.org/mediawiki/2020/d/d3/T--UCL--Poster_idea_section.png"> | |
− | <img src="https://static.igem.org/mediawiki/2020/ | + | |
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<div class="info"> | <div class="info"> | ||
− | + | <div class="title"> | |
− | + | <h1>Idea</h1> | |
− | <div class="title">Idea </ | + | </div> |
− | < | + | <div class="text"> |
− | <div class="text"> | + | <img src="https://static.igem.org/mediawiki/2020/6/6c/T--UCL--Poster_idea_MDC.png"> |
+ | <br> | ||
+ | We propose a technology that can combine enzymatic plastic degradation with seawater desalination in an MDC using engineered microbes to degrade plastic and generate bioelectricity to drive seawater desalination. Our project employed <i>in silico</i> modelling to investigate the potential of using synthetic biology to integrate enzymatic PET degradation with the MDC technology. In a three-chambered device, electricity is generated by the exoelectrogen, <i>S. Oneidensis</i>, which relies upon lactate as its sole food source. Lactate is produced by the co-culture of PET-degrading <i>E. Coli</i> and <i>P. Putida</i> and diffuses to the anode chamber. The transfer of electrons to the anode creates a potential difference that drives the movement of the ions in the seawater out of the middle chamber through ion exchange membranes. | ||
+ | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
− | + | </div> | |
− | </div> | + | |
− | + | ||
− | <!-- | + | <!--Row 3: Research questions and construct design--> |
<div class="row"> | <div class="row"> | ||
− | + | <div class="section half" id="research"> | |
− | <div class="section | + | <img src="https://static.igem.org/mediawiki/2020/0/05/T--UCL--Poster_research_questions.png"> |
− | <img src="https://static.igem.org/mediawiki/2020/0/ | + | |
− | + | <div class="info"> | |
− | + | <div class="title"> Research Questions</div> | |
− | <div class=" | + | <div class="text"> |
− | + | The research questions our project aimed to answer were: | |
− | + | <ol> | |
− | < | + | <li>Can PET be used as a food source for bacteria to generate electricity?</li> |
− | < | + | <li>Can bacteria produce enough electricity for desalination? </li> |
+ | </ol> | ||
+ | <br> | ||
+ | <img src="https://static.igem.org/mediawiki/2020/2/2c/T--UCL--Poster_PET_co-culture.png"> | ||
</div> | </div> | ||
+ | |||
</div> | </div> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <div class="section half" id="construct"> | ||
+ | <img src="https://static.igem.org/mediawiki/2020/8/8b/T--UCL--Poster_construct_design_section.png"> | ||
+ | |||
+ | <div class="info"> | ||
+ | <div class="title">Construct Design</div> | ||
+ | <div class="text"> | ||
+ | We identified strains to express the PET degradation pathway to produce sufficient lactate to sustain the growth and electricity generation by <i>S. Oneidensis</i>. <i>E. coli</i> was engineered to degrade PET into terephthalic acid (TPA) and ethylene glycol (EG) while <i>P. Putida</i> fully metabolizes TPA to produce lactate for <i>S. Oneidensis</i>. | ||
+ | <br><br> | ||
+ | <img src="https://static.igem.org/mediawiki/2020/c/c1/T--UCL--Poster_PET_degradation_pathway.png"> | ||
+ | <p>Caption</p> | ||
+ | <br> | ||
+ | <b>PET degradation pathway construct design:</b> | ||
+ | <br> | ||
+ | We designed constructs that would allow the selected bacteria to to fully degrade PET in the MDC. Therefore, we had the following objectives: | ||
+ | <ol> | ||
+ | <li>Design constructs to degrade PET in <i>E. coli</i> by expressing the PET-degrading enzymes, PETase and MHETase</li> | ||
+ | <li>Express the TPA transporter and degradation pathway in <i>P. putida</i></li> | ||
+ | </ol> | ||
+ | PETase and MHETase were expressed in E. coli as a fusion protein to increase the efficiency of PET degradation. We made a structural model and consulted experts prior to designing the constructs decide on the gene order of the enzymes in the fusion protein. The model showed that the MHETase-PETase construct had less steric hinderance which we hypothesized to perform better. The McGeehan group from the University of Portsmouth also verified our hypothesis as they found that the PETase-MHETase fusion could not be functionally expressed in <i>E. coli</i> due to protein aggregation (4). Therefore, we designed constructs with the individual enzymes and the two configurations of the fusion protein to test if the gene order affected extracellular secretion, functional expression or enzyme activity. | ||
+ | <br><br> | ||
+ | <img src="https://static.igem.org/mediawiki/2020/3/39/T--UCL--Poster_constructs.png"> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | |||
</div> | </div> | ||
− | + | </div> | |
− | <!-- | + | |
+ | <!--Row 4: Co-culture and current output & desalination--> | ||
<div class="row"> | <div class="row"> | ||
− | + | <div class="section" style="width:37.5%;" id="co-culture"> | |
− | <div class="section | + | <img src="https://static.igem.org/mediawiki/2020/9/9a/T--UCL--Poster_co-culture_section.png"> |
− | + | ||
− | <img src="https://static.igem.org/mediawiki/2020/ | + | |
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<div class="info"> | <div class="info"> | ||
− | < | + | <div class="title">Optimising Co-culture Design</div> |
− | <div class=" | + | <div class="text"> |
− | + | Exoelectrogenic behaviour is exhibited during anaerobic respiration, where oxygen cannot be used as the terminal electron acceptor. We performed a Flux Balance Analysis (FBA) over our proposed two-species metabolic pathway to investigate the growth and lactate production of the co-culture in aerobic and anaerobic conditions. | |
− | + | <br> | |
− | </div> | + | The flux balance analysis (FBA) was performed to: |
+ | <ol> | ||
+ | <li>Model the growth of <i>E. coli</i> and <i>P. putida</i> under different oxygen conditions</li> | ||
+ | <li>Optimise TPA production by <i>E. coli</i> and lactate secretion by <i>P. putida</i></li> | ||
+ | </ol> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2020/3/36/T--UCL--Poster_FBA_results_1.png" style="float: left;"> | ||
+ | <img src="https://static.igem.org/mediawiki/2020/f/f9/T--UCL--Poster_FBA_results_2.png" style="float: left;"> | ||
+ | <p>1. <i>P. putida</i> biomass growth simulation on varying TPA and oxygen uptake rates. 2. <i>P. putida</i> lactate secretion simulation on varying TPA uptake rate and biomass growth rate.</p> | ||
+ | |||
+ | The results show that <i>E. coli</i> and <i>P. putida</i> can only grow and produce sufficient lactate to support the growth of <i>S. oneidensis</i> in aerobic conditions. In aerobic conditions, the maximum lactate secretion rate reached by <i>P. Putida</i> is 8.2132 mmol/gDCW/h at a growth rate of 0.2 h-1. | ||
+ | <i>E. Coli</i> is unable to degrade PET under anaerobic conditions and therefore no TPA would be produced for <i>P. Putida</i>. | ||
+ | </div> | ||
+ | </div> | ||
</div> | </div> | ||
− | + | <div class="section" style="width:61.5%;" id="desalination"> | |
− | <div class="section | + | <img src="https://static.igem.org/mediawiki/2020/2/2a/T--UCL--Poster_current_output_section.png"> |
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− | <img src="https://static.igem.org/mediawiki/2020/ | + | |
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<div class="info"> | <div class="info"> | ||
− | < | + | <div class="title">Current Output and Desalination </div> |
− | <div class=" | + | <div class="text"> |
− | + | We built a cellular automata (CA) to investigate the 2nd research question which modelled the current density generated by the formation of an <i>S. oneidensis</i> biofilm on the anode surface and linked to a desalination rate model. | |
− | + | <br> | |
− | </div> | + | <b>Aims of CA model:</b> |
− | </div> | + | <ul> |
− | + | <li>Investigate the growth of <i>S. oneidensis</i> biofilm on the anode for electricity generation</li> | |
− | + | <li>Predict desalination efficiency achieved over electron transfer models</li> | |
− | <div class="section | + | </ul> |
− | + | <br><br> | |
− | <img src="https://static.igem.org/mediawiki/2020/ | + | <img src="https://static.igem.org/mediawiki/2020/8/8c/T--UCL--Poster_CA_biofilm_results.png"> |
− | + | <img src="https://static.igem.org/mediawiki/2020/3/3b/T--UCL--Poster_CA_current_density_results.png"> | |
− | + | <p>1. Agent type over one a week, with time in a hours, showing active cells in blue, quiescent cells in orange, and dead cells in green. Each agent behaves as 125 cells. 2. Total current density generated by biofilm over one week</p> | |
+ | |||
+ | We observed a peak in the current density after about 48 hours at 1.2 A/m2. A steady state current density of approximately 0.0397 A/m2 was achieved lasting for over 9 days generating an average desalination rate of 0.623 L/m2/h. | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <!--Row 5: Market Research and implementation--> | ||
+ | <div class="row"> | ||
+ | <div class="section half" id="market"> | ||
+ | <img src="https://static.igem.org/mediawiki/2020/c/c8/T--UCL--Poster_market_research_section.png"> | ||
<div class="info"> | <div class="info"> | ||
− | + | <div class="title">Market Research</div> | |
− | <div class="title"> | + | <div class="text"> |
− | < | + | Our project, once implemented in the real world, would be novel pieces of infrastructure constructed at the seaside of the selected markets. We have selected Southern California as our test market while Ghana and Nigeria were selected as the final markets as they are all coastal regions and have been suffering from both plastic pollution and fresh water scarcity. |
− | + | <br> | |
− | + | <img src="market research map.png"> | |
− | + | <br> | |
− | </ | + | <b>Criteria for Market Selection</b> |
− | + | Criteria for market selection: | |
+ | <ul> | ||
+ | <li>Struggles with oceanic plastic pollution</li> | ||
+ | <li>Coastal region with a need of fresh water for irrigation </li> | ||
+ | <li>Strong and active NGO activities to ease up communications as we are aiming at building connections initially with NGOs for advertising of and more general suggestions on our project design, and then penetrate and present the idea to academic and industrial experts for further considerations</li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | <b>Selected Regions:</b> | ||
+ | Target Market: | ||
+ | <ul> | ||
+ | <li>Ghana: We collaborated with the Ashesi iGEM team to gather public opinion on plastic pollution and attitudes towards synthetic biology solutions in Ghana.</li> | ||
+ | <li>Nigeria: We consulted <a href="http://www.recyclepoints.com/">RecyclePoints</a>, an NGO recycling organisation in Nigeria, to gather more information on plastic pollution in Nigeria.</li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | Test Market: | ||
+ | <ul> | ||
+ | <li>Southern California: We communicated with the University of California San Diego, (UCSD) and <a href="https://algalita.org/">Algalita</a>, an NGO focused on plastic pollution in Southern California</li> | ||
+ | </ul> | ||
− | + | </div> | |
− | + | </div> | |
− | < | + | </div> |
− | <div | + | |
− | < | + | <div class="section half" id="implementation"> |
− | <div class="section | + | <img src="https://static.igem.org/mediawiki/2020/b/b2/T--UCL--Poster_implementation_section.png"> |
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− | <img src="https://static.igem.org/mediawiki/2020/b/ | + | |
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<div class="info"> | <div class="info"> | ||
− | + | <div class="title">Implementation</div> | |
− | <div class="title"> | + | <div class="text"> |
− | < | + | <img src="https://static.igem.org/mediawiki/2020/5/50/T--UCL--Poster_implementation_summary.png"> |
− | + | The proposed implementation of our MDC technology would be within an industrial desalination plant to produce fresh water for irrigation. After interacting with stakeholders and experts in the desalination industry, we developed a flowsheet of the steps to be included in the MDC desalination process to ensure a safe and efficient implementation of the technology. | |
+ | <br> | ||
+ | <img src="https://static.igem.org/mediawiki/2020/0/04/T--UCL--Poster_flowchart.png"> | ||
+ | </div> | ||
</div> | </div> | ||
− | |||
</div> | </div> | ||
− | + | </div> | |
− | + | ||
− | <div class="section | + | <!--Row 6: Human Practices and Science Communication--> |
− | < | + | <div class="row"> |
− | <img src="https://static.igem.org/mediawiki/2020/ | + | <div class="section" style="width: 39.5%;" id="hp"> |
− | + | <img src="https://static.igem.org/mediawiki/2020/e/e2/T--UCL--Poster_Human_Practices_section.png"> | |
− | <!-- | + | <div class="info"> |
+ | <div class="title">Human Practices</div> | ||
+ | <div class="text"> | ||
+ | In collaboration with Team Ashesi, we distributed a survey to gather opinions on plastic pollution and attitudes towards genetic engineering solutions in Ghana. Generally, from the results of the surveys and interviews that have been done, most end-users (>90%) considered plastic pollution and water scarcity as severe in their regions and more than 60% of them are willing to have our project launched as a novel solution. | ||
+ | <br> | ||
+ | <img src=""> | ||
+ | 1 indicates extremely negative while 5 indicates extremely positive (rest of figure legend) | ||
+ | <br> | ||
+ | <img src=""> | ||
+ | Legend? | ||
+ | <br> | ||
+ | The survey showed that ~60% of the participants expressed a positive attitude to the use of GMOs to tackle plastic pollution and more than 50% are willing to purchase crops irrigated by our desalinated water. | ||
+ | |||
+ | About 86% of the participants also answered that they would like further information about the safety of GMOs to feel more comfortable about consuming crops treated by the MDC technology. Therefore, we created a brochure with Ashesi to educate the end-users about synthetic biology and how they would benefit from our project. | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="section" style="width: 59.5%;" id="education"> | ||
+ | <img src="https://static.igem.org/mediawiki/2020/d/d9/T--UCL--Poster_education_section.png"> | ||
+ | <div class="info"> | ||
+ | <div class="title">Science Communication</div> | ||
+ | <div class="text"> | ||
+ | <img src="sci comm overview" alt=""> | ||
+ | legend? | ||
+ | As our human practices demonstrated, public perception of new biotechnology is improved with improved education and outreach. Water our MDC produces will be used initially for irrigation, and we felt strongly as a team the importance of improving science communication. | ||
+ | Therefore, we achieved the following: | ||
+ | <ul> | ||
+ | <li>Engaged with high school students – the next generation of scientists and engineers - through organised webinars</li> | ||
+ | <li>Targeted outreach activities to underrepresented students in education</li> | ||
+ | <li>Published a fictional magazine story about a synthetic biology superhero who saves a local community from a crisis using the power of engineered bacteria</li> | ||
+ | <li>Used written communication to convey synthetic biology to the public and the wider iGEM community.</li> | ||
+ | </ul> | ||
+ | |||
+ | <img src=""> | ||
+ | <img src=""> | ||
+ | <img src=""> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <!--Row 7: Acheivements and References--> | ||
+ | <div class="row"> | ||
+ | <div class="section half" id="references"> | ||
+ | <img src="https://static.igem.org/mediawiki/2020/4/4a/T--UCL--Poster_references_and_acknowledgements.png"> | ||
<div class="info"> | <div class="info"> | ||
− | |||
<div class="title">References and Acknowledgements</div> | <div class="title">References and Acknowledgements</div> | ||
− | < | + | <div class="text"> |
− | + | <h2>Acknowledgements</h2> | |
− | </div> | + | <b>Sponsors</b> |
+ | <img src=""> | ||
+ | <img src=""> | ||
+ | <img src=""> | ||
+ | <br> | ||
+ | <b>Team members</b> | ||
+ | Olaide Ibiyemi, Juliette Champaud, Stefan Hristov, Pedro Lovatt Garcia, Daniel Castellano, Anna Su, Li Xu and Oliver Hernandez Fernandez | ||
+ | <br> | ||
+ | <b>Supervisors and Instructors</b> | ||
+ | Dr Stefanie Frank, Dr Kenth Gustafsson, Dr Chris Barnes, Dr Darren Nesbeth, Dr Rana Khalife, Alexander Van de Steen (Postgraduate Research student), Rory Gordon (Masters Research Student) and Neythen Treolar (Postgraduate Research Student). | ||
+ | <br> | ||
+ | Thanks to everyone that shared their expertise and supported throughout the project. | ||
+ | |||
+ | <b>References</b> | ||
+ | <ol> | ||
+ | <li>Center for International Environmental Law. Plastic & Climate | The Hidden Costs of a Plastic Planet [Internet]. 2019 [cited 2020 Oct 25]. Available from: https://www.ciel.org/wp-content/uploads/2019/05/Plastic-and-Climate-Executive-Summary-2019.pdf</li> | ||
+ | <li>United Nations. Water scarcity [Internet]. International Decade for Action “Water for Life” 2005-2015. 2014 [cited 2020 Oct 25]. Available from: https://www.un.org/waterforlifedecade/scarcity.shtml</li> | ||
+ | <li>Eriksen M, Lebreton LCM, Carson HS, Thiel M, Moore CJ, Borerro JC, et al. Plastic Pollution in the World’s Oceans: More than 5 Trillion Plastic Pieces Weighing over 250,000 Tons Afloat at Sea. PLoS One. 2014 Dec 10;9(12).</li> | ||
+ | <li>Knott BC, Erickson E, Allen MD, Gado JE, Graham R, Kearns FL, et al. Characterization and engineering of a two-enzyme system for plastics depolymerization. Proc Natl Acad Sci [Internet]. 2020 Oct 13 [cited 2020 Nov 9];117(41):25476–85. Available from: www.pnas.org/cgi/doi/10.1073/pnas.2006753117</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | </div> | ||
</div> | </div> | ||
− | |||
− | + | <div class="section half" id="achievements"> | |
+ | <img src="https://static.igem.org/mediawiki/2020/4/47/T--UCL--Poster_achievements.png"> | ||
+ | <div class="info"> | ||
+ | <div class="title">Achievements</div> | ||
+ | <div class="text"> | ||
+ | <b>Gold medal criteria</b> | ||
+ | Integrated Human Practices | ||
+ | <ul> | ||
+ | <li>Integrated expert feedback into implementation strategy including implementation flowsheets, seawater and plastic pre-treatments, brine treatment, cell replacement, disinfection, post-treatment and distribution systems</li> | ||
+ | <li>Integrated expert feedback into construct and MDC configuration design</li> | ||
+ | <li>On-site visiting of real-world water treatment facilities </li> | ||
+ | <li>Surveys and interviews targeting end-users </li> | ||
+ | <li>Integrated survey and interview results into project reshape, taking end-users' safety and cost concerns, lack of understanding of GMOs into account, and trying to address by using non-pathogenic microorganisms to mitigate safety issues and recycling brine for bringing down the overall costs</li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | Modelling | ||
+ | <ul> | ||
+ | <li>Developed flux balance analysis and cellular automata models to inform the design and operation of the MDC</li> | ||
+ | <li>FBA provided insights into the co-culture conditions for sufficient lactate production</li> | ||
+ | <li>CA predicted the current output and desalination rate produced by <i>S. oneidensis</i></li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | |||
+ | Science Communication | ||
+ | <ul> | ||
+ | <li>Targeted outreach activities to underrepresented students in light of current racial discrimination events</li> | ||
+ | <li>Fostered an open two-way dialogue on synthetic biology through the use of interactive polls</li> | ||
+ | <li>Made materials accessible to a wider audience by translating to different languages and sharing recordings on Youtube</li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | |||
+ | Partnership | ||
+ | <ul> | ||
+ | <li>Partnered with iGEM Exeter in an attempt to characterise our PETase-MHETase fusion protein construct and perform Flux Balance Analysis modelling on their bacterium’s metabolism to gain critical insight into their synthetic biology approaches</li> | ||
+ | <li>Partnered with iGEM Ashesi to target potential stakeholders to improve the human practices & implementation approaches of our projects</li> | ||
+ | </ul> | ||
+ | <br><br> | ||
+ | |||
+ | <b>Silver medal criteria</b> | ||
+ | Engineering Success | ||
+ | <ul> | ||
+ | <li>Followed the engineering cycle to research, design and build PET-degrading constructs to be expressed in <i>E. coli</i> and <i>P. putida</i></li> | ||
+ | <li>Conducted a thorough literature research and consulted with experts for guidance on construct designs</li> | ||
+ | <li>Performed structural modelling to inform the design on the fusion protein construct design</li> | ||
+ | <li>Designed protocols and experiments to test constructs in the lab</li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | |||
+ | Collaboration | ||
+ | <ul> | ||
+ | <li>Collaborated with iGEM MSP-Maastricht to write a peer-reviewed paper for the iGEM 2020 Proceedings Journal</li> | ||
+ | <li>Collaborated with iGEM Exeter to support them with uploading content to their wiki</li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | |||
+ | Human Practices | ||
+ | <ul> | ||
+ | <li>Conducted a market research to identify regions that would benefit the most from our project and regions which had the required infrastructure to support our MDC operations</li> | ||
+ | <li>Engaged with stakeholders and public in selected market locations to assess the feasibility of our project in these regions</li> | ||
+ | <li>Reflected on feedback from survey and experts to inform project implementation</li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | |||
+ | Implementation | ||
+ | <ul> | ||
+ | <li>Designed an MDC desalination process to generate water for irrigation based on feedback from experts</li> | ||
+ | <li>Considered safety aspects of using and containing engineered microbes – made co-culture auxotrophs to limit survivability outside the lab</li> | ||
+ | <li>Considered additional safety aspects of pre-treatment, waste generation, and brine treatment</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
</div> | </div> | ||
Revision as of 04:02, 10 November 2020
Poster: UCL
Poster Template
This poster template will let you create an interactive poster! The poster is divided in two parts: the visual overview on the left and the documentation on the right. The visual overview is broken down into sections that the user can click on. When a user clicks on a poster section in the visual overview, the documentation on the right will display the text and graphics associated with this section of the poster. You can find the documentation on how to use this template, as well as an example here: https://2020.igem.org/Competition/Deliverables/Poster
Project Abstract
The world’s oceans are suffocating from an annual addition of 8 million tons of plastic which threatens marine ecosystems and exacerbates water scarcity, affecting over 2 billion lives globally. Our project aims to tackle these two global challenges by integrating enzymatic polyethylene terephthalate (PET) degradation into a Microbial Desalination Cell (MDC). The system involves a 2-step process of co-culturing engineered E. coli to express a PETase-MHETase fusion degrading PET and P. putida to achieve further degradation and produce lactate, which then supports the biofilm growth of exoelectrogen, S. oneidensis, generating bioelectricity for desalination. Desalination efficiency was maximised by optimising lactate secretion, co-culture design, and MDC configuration based on the results from our flux balance analysis (FBA) and agent-based model, which simulateding bacterial plastic degradation and bioelectricity production, respectively. Our findings were combined with iInsights for further technical optimisation and feasible implementation at large scales, were obtained through iterative repeated engagement with experts and stakeholders.
Problem
Motivation
8 million tons of plastic waste are disposed in the ocean annually (3). With plastic being a common and non-degradable commodity, its linear life cycle is not sustainable for its use. Therefore, we were inspired by the possibility of synthetic biology to create a circular plastic economy by repurposing plastic waste as a resource for other uses.
Idea
We propose a technology that can combine enzymatic plastic degradation with seawater desalination in an MDC using engineered microbes to degrade plastic and generate bioelectricity to drive seawater desalination. Our project employed in silico modelling to investigate the potential of using synthetic biology to integrate enzymatic PET degradation with the MDC technology. In a three-chambered device, electricity is generated by the exoelectrogen, S. Oneidensis, which relies upon lactate as its sole food source. Lactate is produced by the co-culture of PET-degrading E. Coli and P. Putida and diffuses to the anode chamber. The transfer of electrons to the anode creates a potential difference that drives the movement of the ions in the seawater out of the middle chamber through ion exchange membranes.
- Can PET be used as a food source for bacteria to generate electricity?
- Can bacteria produce enough electricity for desalination?
Caption
PET degradation pathway construct design:
We designed constructs that would allow the selected bacteria to to fully degrade PET in the MDC. Therefore, we had the following objectives:
- Design constructs to degrade PET in E. coli by expressing the PET-degrading enzymes, PETase and MHETase
- Express the TPA transporter and degradation pathway in P. putida
The flux balance analysis (FBA) was performed to:
- Model the growth of E. coli and P. putida under different oxygen conditions
- Optimise TPA production by E. coli and lactate secretion by P. putida
1. P. putida biomass growth simulation on varying TPA and oxygen uptake rates. 2. P. putida lactate secretion simulation on varying TPA uptake rate and biomass growth rate.
The results show that E. coli and P. putida can only grow and produce sufficient lactate to support the growth of S. oneidensis in aerobic conditions. In aerobic conditions, the maximum lactate secretion rate reached by P. Putida is 8.2132 mmol/gDCW/h at a growth rate of 0.2 h-1. E. Coli is unable to degrade PET under anaerobic conditions and therefore no TPA would be produced for P. Putida.Aims of CA model:
- Investigate the growth of S. oneidensis biofilm on the anode for electricity generation
- Predict desalination efficiency achieved over electron transfer models
1. Agent type over one a week, with time in a hours, showing active cells in blue, quiescent cells in orange, and dead cells in green. Each agent behaves as 125 cells. 2. Total current density generated by biofilm over one week
We observed a peak in the current density after about 48 hours at 1.2 A/m2. A steady state current density of approximately 0.0397 A/m2 was achieved lasting for over 9 days generating an average desalination rate of 0.623 L/m2/h.Criteria for Market Selection Criteria for market selection:
- Struggles with oceanic plastic pollution
- Coastal region with a need of fresh water for irrigation
- Strong and active NGO activities to ease up communications as we are aiming at building connections initially with NGOs for advertising of and more general suggestions on our project design, and then penetrate and present the idea to academic and industrial experts for further considerations
Selected Regions: Target Market:
- Ghana: We collaborated with the Ashesi iGEM team to gather public opinion on plastic pollution and attitudes towards synthetic biology solutions in Ghana.
- Nigeria: We consulted RecyclePoints, an NGO recycling organisation in Nigeria, to gather more information on plastic pollution in Nigeria.
Test Market:
- Southern California: We communicated with the University of California San Diego, (UCSD) and Algalita, an NGO focused on plastic pollution in Southern California
1 indicates extremely negative while 5 indicates extremely positive (rest of figure legend)
Legend?
The survey showed that ~60% of the participants expressed a positive attitude to the use of GMOs to tackle plastic pollution and more than 50% are willing to purchase crops irrigated by our desalinated water. About 86% of the participants also answered that they would like further information about the safety of GMOs to feel more comfortable about consuming crops treated by the MDC technology. Therefore, we created a brochure with Ashesi to educate the end-users about synthetic biology and how they would benefit from our project.
- Engaged with high school students – the next generation of scientists and engineers - through organised webinars
- Targeted outreach activities to underrepresented students in education
- Published a fictional magazine story about a synthetic biology superhero who saves a local community from a crisis using the power of engineered bacteria
- Used written communication to convey synthetic biology to the public and the wider iGEM community.
Acknowledgements
SponsorsTeam members Olaide Ibiyemi, Juliette Champaud, Stefan Hristov, Pedro Lovatt Garcia, Daniel Castellano, Anna Su, Li Xu and Oliver Hernandez Fernandez
Supervisors and Instructors Dr Stefanie Frank, Dr Kenth Gustafsson, Dr Chris Barnes, Dr Darren Nesbeth, Dr Rana Khalife, Alexander Van de Steen (Postgraduate Research student), Rory Gordon (Masters Research Student) and Neythen Treolar (Postgraduate Research Student).
Thanks to everyone that shared their expertise and supported throughout the project. References
- Center for International Environmental Law. Plastic & Climate | The Hidden Costs of a Plastic Planet [Internet]. 2019 [cited 2020 Oct 25]. Available from: https://www.ciel.org/wp-content/uploads/2019/05/Plastic-and-Climate-Executive-Summary-2019.pdf
- United Nations. Water scarcity [Internet]. International Decade for Action “Water for Life” 2005-2015. 2014 [cited 2020 Oct 25]. Available from: https://www.un.org/waterforlifedecade/scarcity.shtml
- Eriksen M, Lebreton LCM, Carson HS, Thiel M, Moore CJ, Borerro JC, et al. Plastic Pollution in the World’s Oceans: More than 5 Trillion Plastic Pieces Weighing over 250,000 Tons Afloat at Sea. PLoS One. 2014 Dec 10;9(12).
- Knott BC, Erickson E, Allen MD, Gado JE, Graham R, Kearns FL, et al. Characterization and engineering of a two-enzyme system for plastics depolymerization. Proc Natl Acad Sci [Internet]. 2020 Oct 13 [cited 2020 Nov 9];117(41):25476–85. Available from: www.pnas.org/cgi/doi/10.1073/pnas.2006753117
- Integrated expert feedback into implementation strategy including implementation flowsheets, seawater and plastic pre-treatments, brine treatment, cell replacement, disinfection, post-treatment and distribution systems
- Integrated expert feedback into construct and MDC configuration design
- On-site visiting of real-world water treatment facilities
- Surveys and interviews targeting end-users
- Integrated survey and interview results into project reshape, taking end-users' safety and cost concerns, lack of understanding of GMOs into account, and trying to address by using non-pathogenic microorganisms to mitigate safety issues and recycling brine for bringing down the overall costs
Modelling
- Developed flux balance analysis and cellular automata models to inform the design and operation of the MDC
- FBA provided insights into the co-culture conditions for sufficient lactate production
- CA predicted the current output and desalination rate produced by S. oneidensis
Science Communication
- Targeted outreach activities to underrepresented students in light of current racial discrimination events
- Fostered an open two-way dialogue on synthetic biology through the use of interactive polls
- Made materials accessible to a wider audience by translating to different languages and sharing recordings on Youtube
Partnership
- Partnered with iGEM Exeter in an attempt to characterise our PETase-MHETase fusion protein construct and perform Flux Balance Analysis modelling on their bacterium’s metabolism to gain critical insight into their synthetic biology approaches
- Partnered with iGEM Ashesi to target potential stakeholders to improve the human practices & implementation approaches of our projects
Silver medal criteria Engineering Success
- Followed the engineering cycle to research, design and build PET-degrading constructs to be expressed in E. coli and P. putida
- Conducted a thorough literature research and consulted with experts for guidance on construct designs
- Performed structural modelling to inform the design on the fusion protein construct design
- Designed protocols and experiments to test constructs in the lab
Collaboration
- Collaborated with iGEM MSP-Maastricht to write a peer-reviewed paper for the iGEM 2020 Proceedings Journal
- Collaborated with iGEM Exeter to support them with uploading content to their wiki
Human Practices
- Conducted a market research to identify regions that would benefit the most from our project and regions which had the required infrastructure to support our MDC operations
- Engaged with stakeholders and public in selected market locations to assess the feasibility of our project in these regions
- Reflected on feedback from survey and experts to inform project implementation
Implementation
- Designed an MDC desalination process to generate water for irrigation based on feedback from experts
- Considered safety aspects of using and containing engineered microbes – made co-culture auxotrophs to limit survivability outside the lab
- Considered additional safety aspects of pre-treatment, waste generation, and brine treatment